The present invention relates to a method, device, and pharmaceutical compositions, for the controlled removal of cells from the surface of viable tissue by continuous local application of a solution containing a proteolytic enzyme and, more particularly, to a method, device and pharmaceutical composition for non-surgical, enzymatic treatment and biopsy of skin lesions. The method and device each includes a uniquely configured and operative applicator including at least one inlet and at least one outlet, each providing a passageway for streaming of a solution therethrough and over a skin portion defined by a skin-facing opening of a treatment zone of a skin portion of a subject. An opening of at least one of the at least one inlet and the at least one outlet through which the solution streams is height adjustable with respect to the skin-facing opening, such that the applicator physically conforms to a non-smooth skin surface of the subject.
Tissues are composed of individual cells and cell groups, embedded in a proteinaceous extracellular matrix. Collagen fibers are the main component of this ubiquitous network, with other proteins such as fibronectin, laminin, elastin and tenascin, providing a mechanism for cell attachment. Cell surface attachment molecules, such as the CAM proteins, allow cells to adhere to the extracellular matrix and to neighboring cells. Thus, the histological integrity of tissues depends on the interaction of many protein and protein-derived molecules.
Enzymes capable of digesting proteins, or proteases, are commonly employed to disrupt the extracellular matrix of tissues or tissue samples in order to separate cells for establishment of primary cell cultures, for example, as described by Ferkushny, R. I., in “Culture of Animal Cells”, p. 108, A R Liss, NY (1983). Proteases used in the isolation of cells for culturing are typically selective in their proteolytic activity or in the method of their application, achieving effective disruption of the matrix and adhesion proteins, yet causing minimal digestion of critical cell components. In the preparation of a primary culture, the tissue is mechanically cut into small (2–3 mm) pieces, these explants washed and gently agitated in an isotonic buttered solution containing a protease, such as trypsin or collagenase, for 30 minutes to several hours at room temperature. This procedure, resulting in suspended, isolated cells, is universal and has been employed for the preparation and propagation of primary cell cultures from a variety of tissues, including skin biopsies, for example, as described by Hybbinette, S., Bostrom, M., and Lindberg, K., in “Enzymatic Disassociation Of Keratinocytes From Human Skin Biopsies For In Vitro Cell Propagation”, Experimental Dermatology, 8, 30–38 (1999).
Proteolytic digestion of skin, achieving complete or partial disruption of the tissue, has been applied, typically as an alternative to mechanical means, in a wide variety of industrial, cosmetic, experimental and clinical processes. These include the depilation of animal hides and pelts, for example, as disclosed in DE Patent Application No. 19519436, Nov. 28, 1996, soothing and promotion of healing of skin lesions such as CO2 laser surgery wounds, for example, as described by Gaspar, L. and Bogdanyi, E., in “Clinical Experience With Enzymes In The Treatment Of Skin Lesions Caused By CO2 Laser Surgery”, Orv. Hetil. 139, 1475–77 (Hungarian) (1998); and decubitus ulcers, for example, as described by Spoelhof, G. D. and Ide, K., in “Pressure Ulcers In Nursing Home Patients”, Am. Fam. Physic. 47, 1207–15 (1993); the debridement of non-viable tissue as in burn eschar, for example, as described by Mekkes, J. R., LePoole, I. C., Das, P. K., Bos, J. D., and Westerhof, H., in “Efficient Debridement Of Necrotic Wounds Using Proteolytic Enzymes Derived From Antarctic Krill”, Wound Repair and Regeneration, 6, 50–57 (1998), removal of fibrinous exudate from sensitive regions, such as the eye, for example, as described by Mullaney, P. B., Wheeler, D. T., and al-Nahdi, T., in “Dissolution Of Pseudophakic Fibrinous Exudates With Intraocular Streptokinase”, Eye, 10, 362–66 (1996); renewal of aging skin by exfoliation, for example, as disclosed in U.S. Pat. No. 5,976,556 to Norton, et al.; removal of lice nits from hair, for example, as disclosed in U.S. Pat. No. 5,935,572 to Sorenson et al.; and the treatment of infectious lesions of the skin such as acne and leprosy, for example, as disclosed in U.S. Pat. No. 5,958,406 to de Faire et al.
Treatment of Skin Lesions
Treatment of skin lesions such as lentigines, melasmas, keratoses, nevi, keloids, hypertrophic scars, psoriasis, and tattoos requires the removal of diseased or abnormal skin cells. Surgical procedures are generally painful and destructive to the healthy, neighboring tissues, resulting in scarring and abnormal pigmentation of the treated areas, for example, as described by Gambichler, T., Senger, E., Rapp, S., Almouti, D., Altmeyer, P., and Hoffman, K., in “Deep Shave Excision Of Macular Melanocyte Nevi”, Dermatol. Surg., July 26 (7), 662–66 (2000), the need for administration of anesthesia, and significant stress trauma to the patient, for example, as described by Augustin, M., Zschocke, I., Godau, N., Buke-Kirschenbaum, A., Peschen, M., Sommer, B., and Sattler, G., in “Skin Surgery Under Local Anesthesia Leads To Stress-induced Alterations Of Psychological, Physical And Immune Functions”, Dermat. Surg., November 25 (11), 868–71 (1999). In addition, surgical excision of certain lesions is often complicated by the presence of more than one aberrant cell type, for example, as described by Crawford, J. B., Howes, E. L. Jr., and Char, D. A., in “Conjunctival Combined Nevi”, Trans-Am. Ophthamol. Soc., 97, 170–83 (1999), and is inappropriate for sensitive and precarious anatomical regions. These disadvantages of surgical excision of skin lesions have led to the development of non-mechanical methods such as electro-cauterization, electro-ablation, cryosurgery with liquid nitrogen and lasers.
One currently widely used technique employs laser energy directed at the skin lesion to cause ablation of the undesired tissue. Laser surgery, as it is known, is less traumatic to adjoining tissue, due to the cauterization effects of the intense energy, and the ability to carefully focus the laser beam, for example, as described by Raulin, C., Schanermark, M. P., Greve, B., and Werner, S., in “Q-switched Ruby Laser Treatment Of Tattoos And Benign Pigmented Skin Lesions, A Critical Review”, Ann. Plast Surg., November 41 (5), 555–65 (1995), producing less pain and scarring than scalpel or razor blade excision techniques. However, there remain the problems of pain and scarring associated with the intense heat required for tissue ablation, poor results with attempts at laser treatment of certain lesions, such as melanocytic and congenital nevi (Raulin, C. et al., same as above) and the importance of removal, rather than ablation of the abnormal tissue for histological analysis to determine the character and extent of the lesion.
Proteolytic Enzymes in Treatment of Skin Lesions
U.S. Pat. No. 4,226,854 to Klein et al.; U.S. Pat. Nos. 5,505,943 and 6,017,531 to Fortney et al.; U.S. Pat. No. 5,106,621 to Rowan et al., and U.S. Pat. No. 5,840,283 to Sorenson et al., teach the use of proteases to achieve removal or permeation of abnormal, devitalized or necrotic skin. Rowan et al. (U.S. Pat. No. 5,106,621) disclose a cysteine protease from pineapple, ananain, in a pharmaceutical preparation for topical application and debridement of burn wounds or ulcerated tissue. Similarly, Fortney et al. (U.S. Pat. Nos. 5,505,943 and 6,017,531) describe the use of the bacterial protease Vibriolysin for debridement of burn eschar and necrotized skin by topical application in a solution or ointment preparation. Sorenson et al. (U.S. Pat. No. 5,840,283) describe the topical use of proteolytic enzymes as permeation facilitators in treatment of diseased nail, claw or hoof tissue. The commercially available ointment Travase™ (U.S. Pat. No, 3,409,719 to Noe et al.) also employs proteolytic activity, found in Bacillus subtilis filtrate, for the debridement of burn eschar and decubitus ulcer tissue. In all these, and other similar methods, the proteolytic activity is directed at the removal of non-vitalized tissue and is achieved by individual topical applications with gauze or sponge, with no provision for the control of levels or duration of enzyme activity, or the collection of cells from the treated lesion.
U.S. Pat. No. 5,958,406 to de Faire et al. describes the use of a crustacean multifunctional protease for treatment and prevention of bacterial, fungal and viral infections, blood clots, cell-adhesion-related disease (such as HIV and auto-immune disorders) and skin lesions and infection (such as acne, pruritis and scars). The protease preparation is administered by various methods: topically, in an aqueous or non-aqueous vehicle; parenterally, orally or in suppositories for systemic applications; ocularly, in drops, ointment or aerosol; and in cutaneous or subcutaneous injection, for skin lesions such as scars, acne and boils. However, no mention is made of control of enzyme activity once applied, or of a means of obtaining cells from the treated tissues.
U.S. Pat. No. 5,976,556 to Norton et al. describes the topical application of proteolytic enzymes for exfoliation of skin, and treatment of abnormal conditions and diseases of the skin such as warts, lentigines, melasmas, acne, psoriasis, etc. Control of enzyme activity is effected by the restriction of enzymes to acid proteases, active in their acidic buffer when applied, and inactivated by slow deacidification caused by the normal epidermal pH regulatory mechanism. No ongoing monitoring or control of enzymatic activity is provided, and no mention is made of obtaining cells from the treated tissue.
U.S. Pat. No. 6,146,626 to Markert at al. describes the preparation of a proteolytic enzyme mixture comprising collagenase and elastase from Clositridium histolyticum, for application in wound healing and obtaining cells from whole tissue or tissue fragments. Conditions for the topical application of the enzyme to burn wounds, and the isolation of cells from a variety of human and other animal tissue are discussed. However, the procedure described relates to preparation of cells for tissue culture from tissue fragments rather than the therapeutic application of cell removal from live tissue. Furthermore, no provision is made for collection of cells from living tissue in situ.
Autolysis of Proteases
Proteolytic enzymes, being proteins, are in themselves substrates for self-digestion, or autolysis, thus limiting the effectiveness of active, protease based preparations. For example, a commercially available serine protease derived from bacteria of genus Bacillus (Subtilisin A., manufactured by Novo Nordisk Bioindustry Japan K.K.) loses its enzymatic activity by about 70%, when it is kept in an aqueous solution at pH 7.0 at 25° C. for 24 hours. Clinical applications employing proteolytic enzymes should provide means of preventing and controlling catalytic inactivation due to autolysis.
Most proteases demonstrate catalytic activity within a defined, and often narrow physico-chemical environment (pH, temperature, ionic strength, solvent polarity, etc.). The specific nature of some proteases may be exploited to prevent autolysis during storage and application of the enzyme, and to allow for the enzymes deactivation after use.
One approach is to store the enzyme in a lyophilized state, to be diluted in an activating buffer of appropriate pH, ionic strength etc. just before or at the time of delivery to the tissue(s) being treated. Enzyme stability is enhanced when dry, but solubility and even dispersal of the enzyme solid in the activating buffer is difficult, resulting in poor control of enzyme activity at point of delivery.
Another method for prolonging and controlling enzyme activity is the separation of enzyme preparations from their activating buffers until use. This separation may be effected physically, storing the enzyme and activating diluent in separate compartments, the enzyme maintained in a stabilized preparation. U.S. Pat. No. 6,228,323 to Asgharian et al. describes a device for storage and delivery of proteolytic enzyme preparations intended for dispensing contact lens cleanser. The enzyme preparations are stabilized by polyols, such as PEG-400, in a concentrated form, and are mixed with the activating diluents, in predetermined ratios, upon dispensing. In U.S. Pat. No. 5,409,546 to Nakagawa et al. a metal chelator is mixed with the stock enzyme preparation, removing the cations necessary for catalytic activity and prolonging shelf life. Introduction of cations in the diluent restores enzymatic activity, also intended for the cleansing of contact lenses.
A similar approach to prevention of autolysis and destabilization of enzyme preparations is described in U.S. Pat. No. 6,117,433 to Edens et al. The effect of polyols on enzyme activity is discussed in detail, as are other enzyme-stabilizing methods such as non-optimal pH, high salt concentrations, etc. The stabilized enzymes, or other biologically active substances, are intended for dispensing with an appropriate amount of activating diluent, for topical application, as in cosmetic preparations, on skin or other external surfaces. No mention is made of removal and retention of cells from treated skin lesions, or of an apparatus for controlled application of a protease solution to a defined and isolated area.
There is thus a widely recognized need for, and it would be highly advantageous to have, a method and device of controlled enzymatic removal and retention of cells from external surfaces of the skin, devoid of the above limitations.